Functional Groups of Diphosphopyridine Nucleotide-linked Isocitrate Dehydrogenase from Bovine Heart

Fan, Chinan C.; Plaut, G.W.E.

doi:10.1016/s0021-9258(19)42397-1

Cited by 13 publications

(3 citation statements)

References 21 publications

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“…However, there is no resemblance between the cysteine (y subunit, peak VII) labeled by the substrate analogue 3-bromo-2-ketoglutarate in the active site of the pig heart NAD enzyme (Saha et al, 1989) and any of the cysteines of the E. coli enzyme, despite the fact that the bacterial enzyme is also inactivated by 3-bromo-2-ketoglutarate (Ehrlich and Colman, unpublished data). Other papers have reported inactivation of the NAD-dependent isocitrate dehydrogenase from pig heart by reaction of its cysteines with iodoacetate (Mauck & Colman, 1976) and from bovine heart by reaction of cysteines with 5,5'-dithiobis(2nitrobenzoate), TV-ethylmaleimide, and />mercuribenzenesulfonate (Fan & Plaut, 1974). However, several cysteines were modified in each case, and they were not identified within peptides.…”

Section: Discussionmentioning

confidence: 94%

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Huang¹,

Colman²

1990

Biochemistry

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Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.

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Section: Discussionmentioning

confidence: 94%

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Huang¹,

Colman²

1990

Biochemistry

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“…In that case also, the inactivation rate was markedly decreased by the combined addition of Mn2+ and isocitrate, but not by NAD+. Furthermore, the bovine heart NAD+-dependent isocitrate dehydrogenase has also been shown to be inactivated by several sulfhydryl reagents, including 5,5/-dithiobis(2-nitrobenzoate), p-mercuribenzenesulfonate, and vV-ethylmaleimide, with isocitrate and Mn2+ decreasing the inactivation rates (Fan & Plaut, 1974). However, in neither of these studies were the cysteine peptides identified.…”

Section: Separation By Hplc Of [Hc\{carboxymethyl)cysteinementioning

confidence: 91%

“…However, in neither of these studies were the cysteine peptides identified. A critical cysteine of NAD+dependent isocitrate dehydrogenase, which is attacked by BrKG and has now been identified, was probably included among those modified in the earlier studies (Fan & Plaut, 1974;Mauck & Colman, 1976).…”

Section: Separation By Hplc Of [Hc\{carboxymethyl)cysteinementioning

confidence: 95%

Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

Saha

Huang²,

Colman³

1989

Biochemistry

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The substrate affinity label 3-bromo-2-ketoglutarate (BrKG) reacts covalently with pig heart NAD+-specific isocitrate dehydrogenase with complete inactivation and incorporation of about 0.8 mol of reagent/mol of average enzyme subunit [Bednar, R.A., Hartman, F.C., & Colman, R.F. (1982) Biochemistry 21, 3681-3689]. Protection against inactivation is provided by isocitrate and Mn2+. We have now identified a critical modified peptide by comparison of the peptides labeled by BrKG at pH 6.1 in the absence and presence of isocitrate and Mn2+. Modified enzyme, isolated from unreacted BrKG, was incubated with [3H]NaBH4 to reduce the keto group of protein-bound 2-ketoglutarate and thereby introduce a radioactive tracer into the modified amino acid. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated by reverse-phase HPLC, first in 0.1% trifluoroacetic acid with a gradient in acetonitrile and then in 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas-phase sequencing gave the modified peptide: Ser-Ala-X-Val-Pro-Val-Asp-Phe-Glu-Glu-Val-Val-Val-Ser-Ser-Asn-Ala-Asp-Gl u-Glu- Asp-Ile-Arg. The corresponding tryptic peptide that was isolated from unmodified enzyme yielded the same sequence except for (carboxymethyl)cysteine at position 3, suggesting that cysteine is the target of 3-bromo-2-ketoglutarate. Pig heart NAD+-dependent isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma) that can be separated by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The peptide modified by 3-bromo-2-ketoglutarate, which is in or near the substrate site, is derived only from the separated gamma subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

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A compllation of amino acid analyses of proteins, polypeptides, and peptides

Kirschenbaum

1977

Analytical Biochemistry

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Functional Groups of Diphosphopyridine Nucleotide-linked Isocitrate Dehydrogenase from Bovine Heart

Cited by 13 publications

References 21 publications

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

A compllation of amino acid analyses of proteins, polypeptides, and peptides

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